An increasing number of commercial products containing active proteins are becoming available. The majority of these products utilize an enzyme, as the active protein. Enzymes are proteins which react with a compound, or substrate, to break down that compound. Enzymes are divided into numerous classes based on the class of substrate they react upon. Each class of enzyme generally catalyzes the severing of different chemical bonds resulting in the specific selection of activity. The lipase class of enzymes are known for their ability to hydrolyze ester bonds created between, but not limited to, hydrocarbons and polyalcohol backbone substrates. Examples of these substrates are mono-, di-, and triglyceride polyglycerol esters. The protease class of enzymes are known for their ability to hydrolyze proteins. Naturally occurring and bio-engineered protease enzymes are incorporated into household cleaning detergents to hydrolyze proteinaceous dirt and stains, into personal care products to remove dirt and dead skin, into oral cleansing products to facilitate plaque removal in the mouth, and into medicines to affect undesired proteins in the body.
It is known that current commercial cleansing products are made more effective by the incorporation of protease enzymes. U.S. Pat. No. 4,261,868 (Hora et al.), U.S. Pat. No. 4,404,115 (Tai), U.S. Pat. No. 4,318,818 (Letton et al.), European Patent Application 130,756 (published Jan. 9, 1985) and U.S. Pat. No. 5,030,378 (Venegas) all disclose the use of protease enzymes in cleansing or detergent products.
It is also realized, however, that many active proteins, including enzymes, are potential antigens, and may cause allergic reactions in humans under certain conditions. The human immune system can produce specific antibodies upon exposure to active proteins. This process of producing specific antibodies is referred to as "immunization" when a clinically beneficial response is obtained. When the response leads to hypersensitivity, however, it is referred to as "sensitization". Allergenic sensitization to active proteins has been observed in environments where humans are regularly exposed to the protein. Such environments include manufacturing facilities, where workers can be exposed to uncontrolled dust or aerosol containing an active protein, or the marketplace, where consumers' repeated use of products containing active proteins has, on occasion, caused an allergic reaction.
Presently, allergic responses to active proteins can be minimized by limiting the selection of those proteins used in products to those of human origin. While this approach minimizes allergenicity problems, it is not a complete solution since it is often not possible to find such an active protein which also has the activity properties desired.
Another way of diminishing allergic response has been to reduce the size of the protein molecules (see JP Patent Publication Number 4,112,753). However, size reduction may also cause a significant reduction in enzyme activity.
A third proposition for decreasing allergenicity is through epitope mapping and alteration of the protein amino acid sequence to deliver a protein with reduced allergenicity. This approach usually requires a large investment of development time and money.
In the medical field, suggestions have been made to diminish the immunogenicity of proteins through yet another method. This method involves attaching unreactive polymers to the protein. U.S. Pat. No. 4,179,337 (Davis, et al.) relates to enzymes coupled to substantially straight chain polyethylene glycol (PEG) or polypropylene glycol (PPG) polymer moieties. While PEG/PPG coupling was found to mitigate the allergenicity of the enzyme, only 15% of the physiological activity was maintained. PCT Application WO 96/17929 (Olsen, et al., published Jun. 13, 1996) relates to the modification of enzymes by conjugating them with suitable polymers. The Olsen application describes modified enzymes which demonstrate a reduction in allergenicity of from 25% to 66% compared to the parent enzyme, while maintaining from 39% to 100% of the activity of the parent.
The U.S. patent application, Ser. No. 08/903,298 discloses the use of enzymes modified by the addition of twin polyethylene glycol polymer moieties to reduce allergenicity while delivering high enzymatic activity. The modified enzyme therein is used in combination with a fibrous substrate in a wipe application. The modified enzymes are not attached to the substrate. Reduced allergenicity is achieved via the modification of the enzyme.
The U.S. patent application, Ser. No. 09/088,912 disclosed polymeric chemical modification of subtilisin enzymes at one or more of three specific epitope regions which was found to mask the immunogenic determinants of the enzyme.
Another approach to reduce the allergenicity of active proteins has been by granulating, coating or dissolving the active proteins to avoid their becoming airborne. U.S. Pat. No. 4,556,554 (Calvo) discloses cosmetic compositions which comprise enzymes which have been immobilized by attachment to particles of polymeric support. The particles with attached enzymes are dispersed in the cosmetic vehicle. Upon application of the vehicle to the skin, the enzyme is released from the support and is therefore reactivated. Methods such as this address consumer exposure to airborne proteins, however they still leave the substantial risks associated with extended tissue contact with the released enzyme which are deposited on the skin.
Canadian Patent 1,229,808, issued Dec. 1, 1987 teach the immobilization of enzymes, specifically .beta.-galactosidase and .beta.-glucosidase, on cellulosic substrates wherein the enzyme is immobilized by absorption into a agarose gel coating the substrate.
UK Patent Application GB 2,240,040, published Jul. 24, 1991 also teaches immobilized enzymes on substrates. Enzymes, therein as covalently bonded to substrates to provide a medicated dressing.
The activity of enzymes used in biological equipment such as biosensors, bioseparators, and bioreactors has been enhanced by the use of site-specific attachment of enzymes to equipment surfaces. See Huang et al., "Improving the Activity of Immobilized Subtilisin by Site-specific Attachment to Surface", Analytical Chemistry, 69(22), Nov. 15, 1997. Huang teaches the immobilization of subtilisin enzymes via mutation of serine249 or serine145 to cysteine, and bonding to silica beads functionalized with amino groups.
It would be highly desirable to develop a composition which would provide improved levels of protein activity while maintaining low allergenic responses from exposure to the active proteins. If this were accomplished it would provide consumers with safer ways to utilize the benefits of protein technology.
It is an object of the present invention to provide a wipe composition which delivers this improved activity while maintaining reduced stimulation of and resulting activation of the immune system.